The poxviridae comprise a large family of complex DNA viruses that replicate in the cytoplasm of vertebrate and invertebrate cells. In humans smallpox was by far the most important poxvirus infection. The causative agent of smallpox is the variola virus, a member of the genus Orthopoxvirus. Vaccinia virus, also a member of the genus Orthopoxvirus in the family of Poxviridae, was used as live vaccine to immunize against smallpox. Successful worldwide vaccination with Vaccinia virus culminated in the eradication of variola virus (The global eradication of smallpox. Final report of the global commission for the certification of smallpox eradication; History of Public Health, No.4, Geneva: World Health Organization, 1980). In the meantime, most of the stocks of infectious variola viruses have been destroyed. However, it can not be excluded that poxviruses inducing smallpox or smallpoxlike diseases might again become a major health problem. Thus, it is necessary to be in a position to produce stable vaccines against poxvirus infections, in particular variola infections, such as vaccines based on vaccinia virus.
In the past vaccinia viruses have also been used to engineer viral vectors for recombinant gene expression and for the potential use as recombinant live vaccines (Mackett, M., Smith, G. L. and Moss, B. [1982] P.N.A.S. USA 79, 7415–7419; Smith, G. L., Mackett, M. and Moss, B. [1984] Biotechnology and Genetic Engineering Reviews 2, 383–407). This entails inter alia DNA sequences (genes), which code for foreign antigens being introduced into the genome of the Vaccinia viruses with the aid of DNA recombination techniques. If the gene is integrated at a site in the viral DNA which is non-essential for the life cycle of the virus, it is possible for the newly produced recombinant Vaccinia virus to be infectious, i.e. the virus is able to infect foreign cells and thus to express the integrated DNA sequence (EP 83286 and EP 110385). The recombinant Vaccinia viruses prepared in this way can be used, on the one hand, as live vaccines for the prophylaxis of infectious diseases and on the other hand, for the preparation of heterologous proteins in eukaryotic cells. Other examples for recombinant vaccinia viruses are viruses harboring therapeutic genes such as suicide genes, ribozyme genes or antisense genes.
Modified Vaccinia virus Ankara (MVA) is known to be exceptionally safe. MVA has been generated by long-term serial passages of the Ankara strain of Vaccinia virus (CVA) on chicken embryo fibroblasts (for review see Mayr, A., Hochstein-Mintzel, V. and Stickl, H. [1975] Infection 3, 6–14; Swiss Patent No. 568, 392). Examples for MVA virus strains deposited in compliance with the requirements of the Budapest Treaty are strains MVA 572 deposited at the European Collection of Animal Cell Cultures (ECACC), Salisbury (UK) with the deposition number ECACC V94012707, MVA 575 deposited under ECACC V00120707 and MVA-BN with the deposition number ECACC V00083008.
MVA is distinguished by its great attenuation that is to say by diminished virulence or infectiosity while maintaining good immunogenicity. The MVA virus has been analyzed to determine alterations in the genome relative to the wild type CVA strain. Six major deletions of genomic DNA (deletion I, II, III, IV, V, and VI) totaling 31,000 base pairs have been identified (Meyer, H., Sutter, G. and Mayr A. [1991] J. Gen. Virol. 72, 1031–1038). The resulting MVA virus became severely host cell restricted to avian cells. Furthermore, MVA is characterized by its extreme attenuation. When tested in a variety of animal models, MVA was proven to be avirulent even in immunosuppressed animals. More importantly, the excellent properties of the MVA strain have been demonstrated in extensive clinical trials (Mayr et al., Zbl. Bakt. Hyg. I, Abt. Org. B 167, 375–390 [1987], Stickl et al., Dtsch. med. Wschr. 99, 2386–2392 [1974]). During these studies in over 120,000 humans, including high-risk patients, no side effects were associated with the use of MVA vaccine. Recombinant MVA useful as vaccines have already been constructed (see, e.g., WO 97/02355) and are used in clinical trials. WO 98/13500 discloses a recombinant MVA containing and capable of expressing DNA sequences encoding dengue virus antigens. The foreign DNA sequences were recombined into the viral DNA at the site of a naturally occurring deletion in the MVA genome.
An MVA strain showing an even stronger attenuation and enhanced safety characteristics is the strain MVA-BN, deposited at the European Collection of Animal Cell Cultures (ECACC), Salisbury, UK with the deposition number V00083008.
Besides vaccinia virus other poxviruses have been used as vectors to deliver genetic information into mammalian cells. In this context reference is made to avipox viruses such as fowlpoxvirus. Fowlpoxviruses containing HIV genes in the genome are disclosed in U.S. Pat. Nos. 5,736,368 and 6,051,410.
Processes for preparing poxvirus containing compositions suitable as vaccines are known to the person skilled in the art (see for example Joklik W. K., Virology (1962),18, 9–18; Richter, K. H., Abhandlungen aus dem Bundesgesundheitsamt (1970), 9, 53–57). The known purification results in aqueous, poxvirus containing solutions or in poxvirus containing sediments. The poxviruses in these solutions and sediments are not stable, i.e. the infectivity of the viruses rapidly decreases. However, it is necessary that a vaccine can be stored and distributed in a stabilized form, especially when the vaccines need to be transported in tropical regions with limited distribution infrastructure. A freeze-dried product can be stored at temperatures in the range from 4° C. to 25° C. This is a clear advantage compared to the standard storage conditions for liquid formulations, which have to be stored below −20° C. (“Cryopreservation and freeze-drying protocols” Day J, McLellan M; Methods in Molecular Biology, 38, 1995, Humana Press).
Processes for the freeze-drying of poxviruses, in particular vaccinia virus, and virus containing compositions and solutions suitable for this purpose are known (Burke et al., Critical Reviews in Therapeutic Drug Carrier Systems (1999), 16, 1–83). In general terms freeze-drying of a vaccine involves freezing of the vaccine containing aqueous formulation suitable for freeze-drying, followed by removing water by sublimation under conditions of reduced pressure and low temperatures and further followed by removal of water by desorption under conditions of reduced pressure and higher temperatures
The known poxvirus-containing formulations for freeze-drying have important disadvantages. Many of the known vaccinia virus containing compositions for freeze-drying contain peptone or haemaccel, which are often of animal origin. However, there are concerns that animal diseases such as BSE could be transmitted from animal to man via animal products such as peptone, gelatine or haemaccel. Moreover, the poxviruses in the known virus containing formulations for freeze-drying have not been purified. Thus, the poxvirus containing compositions for freeze-drying known in the prior art contain inter alia large amounts of proteins derived from the cells of the cell or tissue culture system and from the bovine serum used during cell cultivation, respectively.
The person skilled in the art also knows freeze-drying compositions that do not contain additional compounds of animal origin (which are e.g. peptone or haemaccel). In this case the compositions contain the following compounds alone or in certain combinations: sodium glutamate, sorbitol, lactose, salts, amino acids and glycerin. However, the product obtained after the freeze-drying process is often rather unstable, i.e. the overall loss in virus titer is unacceptably high during storage. Moreover, it has been shown that the poxvirus tends to form aggregates in some of these formulations and that other compounds precipitate before or during freezing.
U.S. Pat. No. 3,577,526 discloses a smallpox vaccine characterized by the fact that it is made up of a ground virus material of the vaccine dispersed in sucrose. The amount of sucrose is in the range of 20 to 40%. The formulation may further comprise 5% dextran. The term ground virus refers to virus derived from pulps and pustules. Basically, the lymph is ground to break up lumps and separate the liquid from the dead hair and skin. Thus, the protein load of the vaccine preparation is very high and contributes to the stabilization of the virus.